published_or_final_version / Community Medicine / Master / Master of Public Health

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Microarray analysis of gene expression in osteoblasts from patients with osteoporosis (OP) and osteoarthritis (OA) showed that 115 genes were robustly differentially expressed (P<0.05). Functional annotation clustering revealed cell adhesion to be the gene ontology classification most likely to be associated with this gene list. In addition scrutiny of the list revealed several genes with strong biological support for the involvement in bone homeostasis (FOSL1, BMPR2 and TGFBR1). Real -time PCR validated the trends seen in the microarray analysis, but failed to reach statistical significance for any of the genes examined. This analysis supports the value and potential of larger scale comparison of gene expression in OA and OP osteoblasts as a method for identifying novel factors involved in bone homeostasis. The cannabinoid system has recently been identified as involved in the regulation of bone homeostasis. In vitro investigation revealed that although cannnabinoid receptor agonists N-arachidonoylethanolamine (AEA), 2-arachidonoylglycerol (2-AG) and JWH015 had no effect on metabolic activity, cell number, or alkaline phosphatase activity of calvarial mouse osteoblasts there were changes in gene expression. RankL expression was reduced relative to Opg expression by both JWH015 and AEA. Preliminary results indicate that JWH015 was also capable of increasing PPARγ expression which could alter the balance of osteoblastic and adipocytic differentiation of mesenchymal stem cells (MSC). This could have implications for use of these drugs in vivo. Using the 3T3-F442A cell line to develop a model of MSC differentiation highlighted difficulties associated with using cell models. Necessary additional factors required to induce differentiation of a cell line compared to a primary cell make interpretation of results more complicated. This model also highlighted that alkaline phosphatase and osteocalcin (markers usually used to identify osteogenic differentiation) were expressed during adipocytic differentiation. Future use of such markers in MSC models should be closely scrutinized.

Background: To describe the scoring methodology and MRI assessments used to evaluate the cross-sectional features observed in cases and controls, to define change over time for different MRI features, and to report the extent of changes over a 24-month period in the Foundation for National Institutes of Health Osteoarthritis Biomarkers Consortium study nested within the larger Osteoarthritis Initiative (OAI) Study. Methods: We conducted a nested case-control study. Cases (n = 406) were knees having both radiographic and pain progression. Controls (n = 194) were knee osteoarthritis subjects who did not meet the case definition. Groups were matched for Kellgren-Lawrence grade and body mass index. MRIs were acquired using 3 T MRI systems and assessed using the semi-quantitative MOAKS system. MRIs were read at baseline and 24 months for cartilage damage, bone marrow lesions (BML), osteophytes, meniscal damage and extrusion, and Hoffa- and effusion-synovitis. We provide the definition and distribution of change in these biomarkers over time. Results: Seventy-three percent of the cases had subregions with BML worsening (vs. 66 % in controls) (p = 0.102). Little change in osteophytes was seen over 24 months. Twenty-eight percent of cases and 10 % of controls had worsening in meniscal scores in at least one subregion (p < 0.001). Seventy-three percent of cases and 53 % of controls had at least one area with worsening in cartilage surface area (p < 0.001). More cases experienced worsening in Hoffa- and effusion synovitis than controls (17 % vs. 6 % (p < 0.001); 41 % vs. 18 % (p < 0.001), respectively). Conclusions: A wide range of MRI-detected structural pathologies was present in the FNIH cohort. More severe changes, especially for BMLs, cartilage and meniscal damage, were detected primarily among the case group suggesting that early changes in multiple structural domains are associated with radiographic worsening and symptomatic progression.

OBJECTIVE: To determine the effect of total dietary fat intake on the prevalence of hand osteoarthritis (HOA) utilizing data collected in the Osteoarthritis Initiative (OAI) study cohort. METHODS: This is an observational cross-sectional study. Subjects from the OAI cohort with hand radiographs were analyzed for HOA, defined as a Kellgren-Lawrence score of ≥2 in two or more joints on different fingers. Dietary data and socioeconomic factors were collected from the baseline study visit. Logistic regression analysis assessed the association of total fat intake and disease prevalence. Odds ratios were calculated from the coefficients and confidence intervals were calculated with log-likelihood. RESULTS: HOA was prevalent in 1,106 out of the 2,993 participants (37%). Total fat intake did not show a significant relationship to disease prevalence when adjusted for age, education, income, race, smoking, BMI, prescription NSAID use, calcium intake, protein intake, total calories, saturated/monounsaturated fats, and alcohol consumption. There was a significant association of HOA with age, sex, education, race, total calories, and alcohol intake. Sex was analyzed independently to assess for effect modification, showing an association with age (P<0.01) OR (95%CI) 1.03 (1.02,1.03), race (P<0.01) 1.16 (1.09,1.23), and alcohol consumption (P<0.01) 1.06 (1.02,1.09) among only female subjects. Male subjects showed a strong association with age (P<0.01) 1.02 (1.01,1.02). CONCLUSION: Total fat intake does not show a significant association with HOA prevalence with the study sample from the OAI cohort. Age, race, and alcohol consumption showed significant associations depending on sex. More research is needed to further investigation these associations among different groups.

Osteoarthritis (OA) is a chronic, degenerative condition of articular joints. The prevalence of OA is high in many mammalian populations, though our understanding of the disease is limited, with the initiating factors and the early phenotype of the disease being poorly characterised. Clinically, the early-stage of OA is rarely identified, precluding the identification and treatment of affected individuals. Consequently, in vitro models of OA typically reflect the later stages of the disease, and are rarely validated against the naturally-occurring disease. This project utilised tissue from a naturally-occurring canine disease (medial coronoid process disease) to characterise the transcriptome of early-stage OA, and inform different in vitro models, to try and refine the model conditions. Medial coronoid processes from affected dogs were removed and graded histologically, both manually and through the development of a semi-automated assessment. Early-stage OA was characterised by a decrease in the chondrocyte density, an increase in the thickness of the articular cartilage and a loss of proteoglycan. No histological changes in bone morphology were noted in early-stage OA. A transcriptomic approach was adopted, in which the transcriptome of earlystage canine OA was assessed in the coronoid process samples. The canine data generated were meta-analysed alongside published datasets from in vivo models of early-stage OA. These data were from rodent models of the disease. A panel of genes were identified as being associated with the early stage of the disease across multiple datasets. By immunoassay, synovial fluid was screened for pro-inflammatory cytokines and in affected canine joints, interleukin 8 was found to be increased. Three in vitro models (cytokine stimulation of monolayer cell cultures, cyclic compression of agarose embedded cells and impact loading of osteochondral cores) were refined through modification of their stimuli. An identified panel of differentially expressed genes were used to screen each model under different parameters. Hierarchical clustering analysis was used to cluster the panel of conditions so that those which most closely reflected the naturally occurring disease were selected for more detailed transcriptomic analysis by microarray. Chondrocytes and osteoblasts were stimulated with a range of cytokine conditions, using IL-1β and IL-8 based on use in the literature and immunoassay findings. Monolayers were stimulated for a range of times and conentrations with either a single stimulus or multiple cytokines in the medium. The cells responded differently to the cytokine stimulus, requiring different stimuli to most closely replicate the transcriptomic profile of the natural disease. Microarray profiling revealed that cytokine stimulation enriched genes associated with the extracellular matrix and the extracellular region in both cells types. For the cyclic compression model, cells were embedded in an agarose gel matrix and cyclically compressed for various time periods followed by various incubation periods after compression. Both chondrocytes and osteoblasts responded in a similar manner to the cyclic compression stimulus when a post loading incubation step was included to replicate the transcriptomic profile of the natural disease. Cyclic compression enriched gene clusters associated with response to oxidative stress and the extracellular matrix When osteochondral cores were harvested from joints and impacted to represent a traumatic injury, the model could not replicate the transcriptomic model of the natural disease, although increased sGAG release nitric oxide (NO) production was observed. Degradation of mRNA in both tissues was a feature of this model regardless of the loading condition, which precluded further analysis by microarray, but highlighted the significant limitations that were associated with this model. None of the three models tested could accurately reflect the transcriptomic changes of the early-stage OA phenotype in cartilage or bone. A unified model, combining cytokine stimulation with cyclic compression drove cells towards the diseased phenotype in bone. Inflammatory pathways were activated as well as the proteases MMP3 and MMP13. However, chondrocytes were seemingly unresponsive to the multifactorial model, and this will require further analysis. The chronic nature of OA makes it difficult to match in vitro models to the transcriptomic phenotype identified in naturally occurring OA, particularly with respect to the differential expression of structural genes which were identified in the naturally occurring disease but not the models. This work highlights the limitations of existing models, but proposes a validation process which can be used to direct invitro models towards the naturally occurring phenotype.

Osteoarthritis (OA) is a debilitating disease that affects millions of people. It is characterized by the degeneration of articular cartilage, narrowing of the joint cavity, damage to the subchondral bone, loss of synovial fluid, and osteophyte formation. These symptoms can cause muscle weakness, decrease in range of motion at the joint, and pain. Pain is the symptom that is most frequently treated. However, pinpointing the exact origin and location that is causative to the pain can be difficult. Patients of OA commonly have areas of subchondral edema identified on an MRI as marrow bone lesions (BML). On a CT image, these BML are found to be areas of bone resorption within the subchondral bone of the affected joint called subchondral bone cysts (SBC). These cysts are hypothesized to be a source of pain in OA as well as progression of the disease. The synovial intrusion theory for cyst formation states that there is a physical connection made between the joint cavity and the SBC through which synovial fluid travels. The bony contusion theory describes micro cracks developing below the articular cartilage within the subchondral bone causing bone necrosis and SBC formation. This study was undertaken to investigate the histological presentation of the contents of the SBC and the surrounding area.Seven human femoral head samples, ranging from age 49-72 years old, from patients who received total hip arthroplasty due to OA were examined. Three areas were sectioned from each femoral head including an area containing a cyst, an area of primary compressive bone, and an area at the medial side of the femoral head. These sections were then stained for bone, cartilage, and nerves and examined histologically. Sclerotic bone was shown to surround each cyst cavity, while cysts were composed of a mixed connective tissue infiltrated with multiple blood vessels and potential nerve fibers. With further investigation of these structures, the location of the nerve fibers within the SBC could be a possible source of pain in OA and a target for future treatments and therapies. Within the femoral head, cysts were found to be both shallow and deep to the articular cartilage. Shallow cysts may support the synovial intrusion theory for cyst formation while deep cysts could support the bony contusion theory. In relation to articular cartilage, cysts were not only found below a degraded articular cartilage surface as expected but also below intact, less degraded cartilage. This in depth look at the cells and tissues present within and surrounding the cyst provides information to better understand the pathology of OA and possibly an alternative method for treating the disease.

In klinischen Studien wurden bereits positive Effekte des standardisierten Kiefernrindenextrakts Pycnogenol® auf die Symptome von Patienten mit milden Formen von Kniegelenks-Osteoarthritis ermittelt; hauptsächlich ausgedrückt durch Senkung des WOMAC-Scores. Der hinter dieser Symptomverbesserung zu Grunde liegende Mechanismus wurde jedoch noch nicht untersucht. Deshalb sollten in der vorliegenden Arbeit erstmalig die zellulären pharmakodynamischen Effekte des Nahrungsergänzungsmittels, in Hinblick auf wichtige Marker der Knorpelhomöostase, untersucht werden. Hierfür wurden 30 Patienten mit schweren Gonarthrose-Formen und Indikation zum Kniegelenksersatz in eine randomisiert-kontrollierte Studie eingeschlossen. Die genaue Ursache der Erkrankung Osteoarthritis ist bis heute nicht geklärt, jedoch gilt ein Ungleichgewicht von Knorpelaufbau und –abbau in den betroffenen Gelenken als einer der zentralen Parameter der Pathogenese. Diese Imbalance resultiert in einem sukzessiven Knorpelverlust, der mit einem Entzündungsgeschehen im ganzen Gelenk, also auch unter Beteiligung von Synovium und subchondralen Knochen, einhergeht. Eine wichtige Rolle spielen hierbei die matrix-abbauenden Enzyme MMPs und ADAMTS sowie proinflammatorische Mediatoren, z.B. das IL-1β. Nach dreiwöchiger Einnahme von 200 mg Pycnogenol® am Tag, konnten wir, im Vergleich zur unbehandelten Kontrollgruppe, eine Senkung der relativen Genexpression von MMP-1, MMP-3 und MMP-13 im Knorpelgewebe feststellen. Bei MMP-3 und MMP 13 war diese Reduktion signifikant. Ebenso wurde die relative Expression von IL-1β statistisch signifikant gesenkt. Im Rahmen der Untersuchung der Entwicklung von Markerkonzentrationen im Serum im Verlauf der Studie wurde eine signifikante Senkung der ADAMTS-5-Konzentrationen bei behandelten Patienten, im Vergleich zur Kontrollgruppe, offenbar. Weiterhin wurden die MMP-13-Konzentrationen im Serum positiv durch Einnahme des Rindenextraktes beeinflusst. In der Körperflüssigkeit, die dem Erkrankungsgeschehen am nähesten kommt, der Synovialflüssigkeit, konnten ebenso hemmende Effekte auf knorpelabbauende Enzyme nach Einnahme von Pycnogenol® beobachtet werden. Hierbei sah man niedrigere Konzentrationen der Marker MMP-1 und MMP-13 sowie der Abbaumarker von Typ-II-Collagen und von Aggrecan in den Gelenkflüssigkeiten der Verum- im Vergleich zu denen der Kontrollgruppe. Im Rahmen von ex-vivo-Versuchen zeigten sich mit beiden Spezimen keine Unterschiede zwischen den beiden Studiengruppen. Die beobachteten Tendenzen konnten durch Korrelationsanalysen untermauert werden. Die Ergebnisse der vorliegenden Arbeit liefern den ersten Ansatz zum Verständnis der zellulären Mechanismen, die für die positiven Einflüsse des standardisierten Kiefernrindenextraktes auf die Symptomatik der Gonarthrose verantwortlich sind. Weitere Studien mit einer größeren Studienpopulation und einer Anwendung von Pycnogenol® über einen längeren Zeitraum sind nötig, um diese zellulären Geschehnisse zu bestätigen und näher zu untersuchen. Auf Grund des günstigen Nebenwirkungsprofils von Pycnogenol® ist eine Langzeittherapie zur Verzögerung eines erstmaligen Kniegelenksersatzes durchaus denkbar. Dies hätte den Vorteil, dass das betroffene Gelenk weniger oft ausgetauscht werden müsste, was wegen der begrenzten Haltbarkeit in etwa alle 10 Jahre geschieht. Aus epidemiologischen Studien ist schon seit Längerem bekannt, dass eine hohe tägliche Aufnahme von Polyphenolen über die Nahrung zu geringeren Inzidenzraten neurologischer Erkrankungen, wie z.B. Morbus Parkinson oder Morbus Alzheimer, führt. Auch Pycnogenol® hat in-vivo schon positive Effekte auf diverse neurologische Erkrankungsgeschehen gezeigt. Um zu verstehen, welcher Inhaltsstoff bzw. welche Inhaltsstoffe und/oder Metabolite die Blut-Hirn-Schranke passieren und für diese Wirkungen verantwortlich sein könnten, wurde in der vorliegenden Arbeit mit Hilfe eines cEND-in-vitro-Modells die Blut-Hirn-Schrankengängigkeit ausgewählter Bestandteile des Extraktes und des Metaboliten M1 untersucht. Dabei zeigte keine der untersuchten Substanzen unter den gewählten Versuchsbedingungen einen quantifizierbaren Übertritt durch den Zellkultur-Monolayer. Auf Grund unserer Versuche ist jedoch eine Aufnahme des M1 und von (+)-Catechin in die Endothelzellen durchaus denkbar. Diese Aufnahme scheint für den M1, in erleichterter Form, durch den GLUT-1-Transporter zu verlaufen. Die positiven Effekte des Nahrungsergänzungsmittels auf neurologische Erkrankungen scheinen nicht durch direkte Einwirkungen im Gehirn selbst verursacht zu werden. Eine stabilisierende Wirkung auf die BHS, die eine wichtige Barriere zum Schutz des Gehirns vor äußeren Einflüssen ist, scheint dafür eine plausiblere Erklärung zu sein. Weiterführende in-vivo-Tierversuche können darüber Aufschluss geben. Zusammenfassend konnte mit der vorliegenden Arbeit ein Beitrag zur Aufklärung der zellulären Effekte des standardisierten Kiefernrindenextraktes bei schwerer Kniegelenks-Osteoarthritis geleistet werden. Zusätzlich konnten wir, mit Hilfe eines rationalen Ansatzes zur Ermittlung der Blut-Hirn-Schrankengängigkeit ausgewählter Inhaltsstoffe von Pycnogenol®, das Verständnis für die positiven Wirkungen von Pycnogenol® im Rahmen neurologischer Erkrankungen erweitern. / In clinical trials, positive effects of standardized pine bark extract (Pycnogenol®) on symptoms of patients with mild forms of knee osteoarthritis were already seen, mostly identified by reduction of WOMAC scores. The underlying mechanisms were not investigated so far. Because of that, in the present work the cellular pharmacodynamic effects of the dietary supplement Pycnogenol® with regard to important markers of cartilage homeostasis should be observed. Therefore, 30 patients with severe forms of knee osteoarthritis, who had an indication for knee replacement surgery, were included. The precise cause of osteoarthritis is so far unknown, but an imbalance of buildup and depletion of cartilage in affected joints is considered to be a hallmark of pathogenesis. This imbalance results in successive loss of tissue, which goes along with inflammatory processes in the whole joint, also affecting synovium and subchondral bone. Here, matrix-degrading enzymes like MMPs and ADAMTS, as well as inflammatory mediators, e.g. IL-1β, play important roles. After daily intake of 200 mg Pycnogenol® over three weeks, reductions of relative gene expressions of MMP-1, MMP-3 and MMP-13 were observed. Regarding MMP-3 and MMP-13, this reduction was statistically significant. Relative gene expression of IL-1β was also diminished significantly. In context of the investigation of marker concentration developments in serum we observed a statistically significant reduction of ADAMTS-5 concentrations during the clinical trial in treated patients in relation to controls. Furthermore, MMP-13 concentrations were positively influenced by oral intake of pine bark extract. Regarding synovial fluid, the body fluid next to disease events, we also determined modulating effects through intake of Pycnogenol®. Here we could observe lower levels of MMP-1 and MMP-13, as well as of markers of degradation of aggrecan and type II collagen, in joint fluids of patients who took Pycnogenol® in relation to control group. In context of ex-vivo-approaches, including both kinds of samples, no differences were observed between the two study groups. The observed tendencies could be confirmed by correlation analysis. The results of the present work provide a first approach to understand the cellular mechanisms, which are responsible for the positive influences of standardized pine bark extract on symptoms of gonarthrosis. More clinical trials with greater study populations and longer intake of Pycnogenol® are needed to confirm the observed cellular effects and to investigate them in more detail. Because of good side effect profile, long-term use of pine bark extract for delaying the date of knee replacement surgery seems possible. Renewing affected joints less often, which takes place around every ten years, would be the advantage of this time lag. From epidemiological studies we know for quite some time that high daily intakes of polyphenols via food result in lower incidence rates of neurological disorders, like e.g. Parkinson’s disease and Alzheimer’s disease. Pycnogenol® also has already shown positive effects on neurological disturbances in-vitro and in-vivo. To understand, which ingredient or which ingredients and/or metabolites, respectively, are responsible for these effects, we measured the blood-brain barrier permeability of selected components of Pycnogenol® and its metabolite M1 in the present work by means of a cEND in-vitro model of this barrier. None of the investigated substances showed a quantifiable transfer through cell culture monolayers under present conditions. On basis of our approaches, an uptake of M1 and (+)-catechine in endothelial cells is reasonable, however. In this context, a facilitated uptake of M1 through GLUT-1 transporters seems likely. Positive influences of the dietary supplement on neurological disorders seem not to be caused by direct effects in brain. A stabilizing impact on the blood-brain barrier, which protects the brain from external influences, could be a more likely explanation. Further in-vivo animal studies possibly could shed more light on this issue. In summary the present work could make a contribution to the elucidation of the cellular mechanisms of standardized maritime pine bark extract in severe gonarthrosis of the knee. Additionally we could enlarge, by means of a rational approach to determine the blood-brain barrier permeability of selected ingredients of Pycnogenol®, the understanding of the positive impacts of Pycnogenol® in neurological disorders.

Doctor of Philosophy / The aim of the studies reported in this project was to examine factors associated with the prevention and management of trapeziometacarpal osteoarthritis, both in musculoskeletal physiotherapists and the general patient population. Two studies were undertaken to investigate factors associated with the aggravation of thumb pain in musculoskeletal physiotherapists. Study 1 was a survey of the prevalence of thumb pain, and allowed determination of the most aggravating spinal manipulative therapy technique. It identified that 83% of respondents complained of an aggravation of thumb pain due to the performance of spinal manipulative therapy techniques, with 85-87% of the painful respondents complaining of thumb pain aggravated by unilateral and central PA glides. Study 2 was conducted to determine whether the alignment of the joints of the thumb during performance of these glides was associated with thumb pain. This observational study of 129 musculoskeletal physiotherapists performing a PA glide identified that aligning the metacarpophalangeal and interphalangeal joints in extension was associated with a lower prevalence of work-related thumb pain. Therefore, it is suggested that musculoskeletal physiotherapists be taught to perform these techniques with the joints of their thumb in extension in an effort to reduce the development of work-related thumb pain. Furthermore, it is suggested that those who are unable to maintain this alignment voluntarily be provided with a thermoplastic thumb splint to maintain the extended alignment. Two studies were undertaken to investigate the conservative and surgical management of patients with trapeziometacarpal osteoarthritis. Study 3 was a randomised controlled trial conducted to compare the efficacy of a new thumb strap splint and an abduction exercise regimen against the standard approach to conservative management of trapeziometacarpal osteoarthritis, namely a short opponens splint and pinch exercise regimen. While there was no additional benefit of one approach over the other, all participants improved in the outcomes of pain, strength and hand function over the six-week period of intervention. Nevertheless, some people find that symptom relief from conservative intervention is inadequate and short-lived, requesting surgery for the treatment of disabling and persistent pain from trapeziometacarpal osteoarthritis. Study 4 was a systematic review, conducted to determine evidence of efficacy of one surgical procedure over another. This review identified six randomised controlled trials of surgery for trapeziometacarpal osteoarthritis. While there was evidence of no difference in the reduction in weakness between the procedures, there was insufficient evidence to confirm that there was no difference in the outcomes of pain, contracture, hand function, or patient global assessment. Furthermore, there was sufficient evidence to conclude that trapeziectomy had significantly fewer adverse effects, and trapeziectomy with ligament reconstruction and tendon interposition (LRTI) had significantly more, when compared with the other procedures analysed in this review. It is suggested that the decision as to which intervention is most appropriate for a given patient be based upon the individual patient’s requirements, the extent of disease, and the demands placed upon the joint by domestic duties, work, leisure and recreational activities. The studies presented in this project assist in formulating preventative and management strategies for people with trapeziometacarpal osteoarthritis.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 65-70). Also available on the Internet.